Background and Hypothesis: Prostate cancer (PCa) remains the second leading cause of cancer death in men in the United States. During the previous funding period, we have found that phenethyl isothiocyanate (PEITC) and curcumin (CUR) alone and in combination inhibit the development and progression of PCa in nude mice PC-3 xenograft and TRAMP mice via induction of pro-apoptotic and anti-proliferative signaling pathways. The objective of this R01 continuation application is to investigate the epigenetic effects of PEITC against PCa. Rationale for the studies proposed in this application is derived from our published and unpublished preliminary studies showing that: (i) As TRAMP PCa progresses, there is a progressive loss of the anti-oxidative stress transcription factor Nrf2 and Nrf2-target genes phase II detoxifying and antioxidant enzymes;(ii) Treatment with PEITC resulted in the re-expression of Nrf2 and Nrf2-target gene UGT1A1 in the TRAMP prostate tumor correlating with tumor suppression;(iii) Nrf2 gene is epigenetically regulated in TRAMP tumor and TRAMP C1 cell line through promoter CpG hypermethylation;(iv) ChIP assays revealed increased binding of the MBD2 and trimethyl-histone H3 (Lys9) proteins to these CpG sites;(v) treatment with DNMT inhibitor 5-aza and HDACi TSA restores the expression of Nrf2;(vi) PEITC reverses the methylation status of certain genes in LNCaP cells from a panel of 96 gene promoters using DNA methylation PCR Array;(vii) PEITC restores expression of 14-3-3 gene by MSP assay and qPCR. Despite these promising results, significant gaps exist in our understanding of the epigenetic mechanism(s) of PEITC. Based on the results of our preliminary studies we would like to test our hypothesis that epigenetic events would drive the development and progression of PCa in TRAMP and PEITC modifies these alterations leading to its PCa chemopreventive effect. The epigenetic events of Nrf2 and Nrf2-target genes in TRAMP as well as pro- apoptotic/anti-proliferative genes in LNCaP will be investigated in detailed both in vivo and in vitro. Specific Aims: The specific aims of this project are to: (1) To investigate the epigenetic alterations during the development and progression of prostate tumorigenesis in TRAMP mice and the chemopreventive effects of PEITC through epigenetic modifications. (2) To determine the epigenetic modifications of pro-apoptotic/anti- proliferative genes elicited by dietary PEITC on the growth inhibition of LNCaP orthotopic xenograft tumors in SCID mice. (3) To elucidate the in vitro epigenetic mechanisms of regulation of the genes obtained from in vivo Aims 1 and 2 by PEITC in TRAMP C1, C3 and LNCaP cell lines. Significance of the Proposed Research: Cancer chemoprevention of PCa through dietary or chemical intervention would be logical and would be tremendous clinical benefit to hundreds of thousands of men in the United States. The success of the proposed study will further drive clinical trials of PEITC to determine its chemopreventive epigenetic mechanisms, biomarkers and effectiveness against human PCa. PUBLIC HEALTH RELEVANCE: Prostate cancer (PCa) remains one of the deadliest cancers in men in the US. The long term goal of this application is to develop an effective and safe strategy for prevention of PCa in men using a natural dietary compound, phenethyl isothiocyanate (PEITC) found abundantly in garden cress via epigenetic modification.